I.
Gastric
cannulation
a.
Mice
are
fasted
for
24h
and
then
anesthetized
for
surgery
by
inhalation
of
methoxyflurane.
b.
While
adhering
to
aseptic
surgical
techniques,
a
45-cm
PE-90
polyethylene
tube
with
a
small
silicon
tip
(1.5
mm)
on
one
end
is
used
for
gastric
cannulation.c.
Once
fully
anesthetized,
a
vertical
midline
incision
is
made
in
the
skin
of
the
abdomen
from
the
xiphoid
cartilage
extending
to
the
midabdomen
near
the
umbilicus.d.
A
second
small
incision
is
made
in
the
dorsal
cervical
region
between
the
scapular
bones.
e.
A
subcutaneous
tunnel
is
exposed,
and
7-0
polypropylene
sutures
are
passed
1-mm
apart
through
the
serosa
and
muscular
layer
of
the
stomach.
f.
After
a
small
opening
is
made
in
the
forestomach
between
sutures,
the
proximal
tip
of
the
cannula
is
inserted
0.8
cm
into
the
stomach.g.
The
tube
is
anchored
to
the
stomach
wall
with
Dacron®
suture
and
the
stomach
is
replaced
back
into
the
abdominal
cavity.
h.
The
small
incision
where
the
cannula
exited
through
the
abdominal
wall
is
closed
with
5-0
silk
and
tied
around
the
cannula,
resulting
in
tight
fixation
of
the
cannula
to
the
abdominal
wall.
i.
The
abdominal
wall
and
skin
are
closed
and
sutured
with
5-0
silk
suture.
j.
Mice
are
placed
in
a
prone
position
and
an
anchoring
button
is
sutured
to
the
muscles
of
the
dorsal
cervix,
and
the
skin
is
closed
around
the
button
stem
with
5-0
silk
suture.
The
estimated
duration
for
the
total
surgical
procedure
is
~30
min.
k.
To
prevent
postsurgical
infection,
gentamicin
(8
mg/kg)
is
administered
intraperitoneally.
l.
The
distal
end
of
the
cannula
is
allowed
to
exit
through
a
flanged
button.
m.
A
protective
spring
coil
encapsulating
the
cannula
is
connected
to
a
swivel,
and
then
anchored
with
a
button,
allowing
the
mice
to
be
able
to
move
freely.n.
Intubated
mice
are
placed
in
metabolic
cages
(singly
housed)
and
the
distal
end
of
the
gastric
cannula
is
connected
to
another
swivel
and
infusion
pump
(Fig.
1).0.
Mice
are
allowed
to
recover
1
wk
with
ad
libitum
access
to
food
and
water.

Figure
1.
Gastric
infusion
model
with
mouse
fitted
with
a
feeding
tube
and
kept
in
a
metabolic
chamber.

II.
Feeding
liquid
high-fat
diet
with
or
without
EtOH,
and
urine
collectiona.
Mice
are
grouped
into
two
cohorts:
(1)
control
group,
high-fat
diet
(HFD)
and
(2)
treatment
group,
high-fat
diet
containing
EtOH
(HFD_EtOH)
continuously
for
up
to
4
weeks
via
intragastric
feeding.
The
two
cohorts
are
given
an
equivalent
amount
of
daily
calories.b.
Liquid
HFD
(1.29
to
1.31
kcal/mL),
supplemented
with
isocaloric
maltose-dextrin
and
lipotropes,
is
infused
at
a
rate
of
0.44
mL/g
body
weight
per
day
with
a
peristaltic
pump.c.
Liquid
HFD_EtOH
is
delivered
initially
at
17.3
g/kg/day
and
is
gradually
increased
1.3
g/kg
every
2
days
until
day
8,
and
then
to
1.2
g/kg
every
4
days
until
the
dose
reaches
27
g/kg/day
(EtOH=40%
of
total
calories).
The
amount
of
EtOH
in
the
diet
is
varied
from
4.0%
to
8.0%
to
obtain
optimal
delivery
of
calories
without
compromising
growth
or
survival.
In
addition
to
the
liquid
diet,
the
mice
are
given
free
access
to
water.d.
Urine
samples
are
collected
daily
from
the
metabolic
cage
collection
cups
and
stored
at
-80°C
until
analysis.

III.
Euthanasia,
blood
collection
via
vena
cava
and
liver
dissection
and
fixationa.
Before
mice
are
euthanized,
final
body
weight
is
obtained.
b.
The
mice
are
euthanized
by
fully
trained
personnel
using
CO2
gas
asphyxiation.c.
Terminal
blood
samples
are
collected
via
the
vena
cava
for
plasma
and
serum
preparation
(see
below).d.
Livers
are
excised
and
weighed.
e.
A
slice
of
the
medial
lobe
(Fig.
2)
is
fixed
in
10%
neutral
buffered
formalin
for
48h
for
histopathologic
examination.
The
remaining
liver
is
snap-frozen
in
liquid
nitrogen
and
stored
at
-80°C
for
protein
extraction
and
RNA
isolation.

Figure
2.
Liver
lobes
and
their
orientation
within
the
abdominal
cavity
viewed
ventro-dorsally.

IV.
Blood
chemistry:
Plasma
homocysteinea.
An
aliquot
(~
450
µL)
of
terminal
blood
is
collected
into
EDTA-containing
tubes
and
immediately
centrifuged
at
1,500
rpm
for
15
min
at
4°C.b.
Aliquots
of
the
plasma
layer
are
transferred
into
cryostat
tubes
and
stored
at
-20°C
until
analysis.
c.
To
determine
the
free
(non-protein
bound)
aminothiol
levels,
an
equal
volume
(100
µL)
of
freshly
prepared
10%
meta-phosphoric
acid
is
added
directly
to
100
µL
plasma
followed
by
a
30-min
incubation
on
ice.
d.
After
centrifugation
for
15
min
at
14,000
rpm
at
4°C,
the
supernatant
is
likewise
filtered
before
injecting
20
µL
of
the
sample
into
the
HPLC
(see
below).

V.
Blood
chemistry:
Serum
amino
transaminase
(ALT)
a.
Blood
samples
are
allowed
to
clot
at
room
temperature
for
at
least
30
min.b.
Serum
is
separated
from
blood
cells
using
a
table
top
centrifuge
at
a
speed
of
14,000
rpm
for
5
min.c.
The
serum
layer
is
pipetted
carefully
without
disturbing
the
packed
blood
cell
layer
below
and
then
transferred
into
a
new
pre-labeled
tube
and
stored
at
-20°C
until
ready
to
be
assayed.d.
An
aliquot
of
frozen
serum
is
completely
thawed
before
testing
for
serum
ALT
levels
using
a
spectrophotometer
with
the
ALT
Liquid
stable
reagent
according
to
manufacturer's
instruction.

VI.
Blood
alcholol
concentration
(BAC)
and
urine
alcohol
concentration
(UAC)EtOH
levels
are
determined
by
measuring
absorbencies
at
340
nm
resulting
from
the
reduction
of
NAD1
to
the
reduced
form
of
nicotinamide
adenine
dinucleotide
by
alcohol
dehydrogenase.a.
Aliquots
of
frozen
serum
and
urine
samples
are
allowed
to
thaw
at
room
temperature.b.
Serum
and
urine
samples
are
first
diluted:
50
µL
of
serum
or
urine
is
added
to
950
µL
of
water.
Volume
can
be
adjusted,
but
the
dilution
factor
is
kept
the
same.c.
Alcohol
standards
are
prepared
by
adding
1.27
mL
of
absolute
EtOH
and
bringing
it
to
100
mL
volume
using
a
volumetric
flask
kept
at
room
temperature
to
make
a
1000
mg/dL
standard.
This
is
diluted
serially
to
get
500,
150,
125
and
0
mg/dL
standards.
Each
standard
is
diluted
the
same
way
as
the
samples
(20X).d.
Reaction
buffer
is
prepared
fresh
and
may
be
stored
with
a
cover
and
refrigerated
for
up
to
48h.e.
In
duplicate,
20
µL
of
sample
or
standards
is
pipetted
into
96-well
plates,
200
µL
of
reaction
buffer
is
added
and
mixed
well.
f.
Plate
is
covered
and
incubated
at
room
temperature
for
1.5
h
before
spectrophotometer
absorbance
is
read
at
340
nm.g.
Standard
curves
are
plotted
and
unknown
values
for
blood
alcohol
concentration
(BAC)
or
urine
alcohol
concentration
(UAC)
are
determined.

VIII.
Liver
protein
and
lipid
extraction
a.
A
50-mg
sample
of
frozen
liver
from
each
mouse
is
homogenized
in
borax/EDTA
(pH
9.3).b.
The
homogenized
liver
is
then
mixed
thoroughly
with
chloroform
by
vortexing
and
then
centrifuging
at
1,500
rpm
for
15
minutes
at
-4°C
to
separate
unwanted
precipitates.c.
For
additional
purification,
liver
samples
are
placed
at
-20°C
for
at
least
2h
followed
by
centrifugation
at
12,000
rpm
at
4°C
for
10
min.d.
Aliquots
of
the
supernatant
are
pipetted
in
duplicate
tubes
per
liver
sample
and
labeled,
one
for
reduced
glutathione
and
the
other
for
total
glutathione,
and
stored
at
-20°C
until
analysis.e.For
determination
of
total
aminothiols,the
disulfide
bonds
are
reduced
and
protein-bound
thiols
are
released
by
the
addition
of
50
µL
freshly
prepared
1.43
M
sodium
borohydride
solution
containing
1.5
µM
EDTA,
66
mM
NaOH,
and
10
µL
n-amyl
alcohol
to
each
aliquot
of
liver
sample.f.
After
gentle
mixing,
the
solution
is
incubated
in
40°C
water
bath
for
30
min
with
gentle
shaking.g.
To
precipitate
and
remove
further
remove
unwanted
liver
proteins,
250
µL
ice-cold
10%
meta-phosphoric
acid
is
added
and
the
sample
is
incubated
for
10
min
on
ice.
h.
After
centrifugation
at
14,000
rpm
for
15
min
at
4°C,
the
supernatant
is
filtered
through
0.2
µ
filter
and
a
20
µL
sample
is
injected
into
the
HPLC
system
described
below.
i.For
the
determination
of
free
(non-protein
bound)
aminothiol
levels,
an
equal
volume
of
freshly
prepared
10%
meta-phosphoric
acid
is
added
directly
to
200
µL
aliquoted
liver
sample
followed
by
a
30-min
incubation
on
ice.
j.
After
centrifugation
for
15
min
at
14,000
rpm
at
4°C,
the
supernatant
is
filtered
as
above
before
injecting
20
µL
of
the
sample
into
the
HPLC
system.
k.
For
the
determination
ofhepatic
triglycerides,
20
mg
of
frozen
liver
tissue
is
homogenized
in
500
µL
of
isopropyl
alcohol,
and
4
µL
of
the
extract
is
used
in
subsequent
analysis.

IX.
Analysis
of
liver
tissue
extracts
and
plasmaPlasma
and
liver
aminothiols
are
separated
by
HPLC
coupled
with
a
Shimadzu
solvent
delivery
system
(model
580).
A
reverse
phase
C18
NBS
column
is
used
with
a
mobile
phase
consisting
of
50
mM
sodium
phosphate
monobasic,
monohydrate,
1.0
mM
ion-pairing
reagent
1-octanesulfonic
acid,
2%
acetonitrile
(v/v),
adjusted
to
pH
2.7
with
85%
phosphoric
acid.a.
Plasma
and
liver
extracts
are
directly
injected
onto
the
column
using
an
autosampler.b.
Isocratic
elution
is
performed
at
ambient
temperature
at
a
flow
rate
of
1.0
mL/min
and
a
pressure
of
120
to
140
kgf/cm2
(1,800
–
2,100
psi;
or
11.7
-
13.7
kPa).
c.
To
assure
standardization
between
sample
runs,
calibration
standards
are
interspersed
at
intervals
and
duplicate
reference
liver
standards
are
included.
Also,
before
sample
analysis,
standard
curves
are
examined
each
day
using
frozen
aliquots
of
stock
calibration
solution.
The
limit
of
detection
for
standard
calibration
standards
is
defined
as
the
concentration
that
produced
a
signal-to-noise
ratio
greater
than
5.d.
Linear
calibration
curves
consisting
of
4
to
5
points
for
each
compound
are
generated
in
the
following
biologic
ranges
for
each
compound:
0.5
to
100
nmol/mL
homocysteine,
5
to
350
nmol/mL
cysteine,
5
to
200
nmol/mL
cysteinylglycine,
0.5
to
100
nmol/mL
reduced
glutathione,
and
0.5
to
500
nmol/mL
oxidized
glutathione.e.
Reduced
liver
glutathione
(GSH),
oxidized
liver
glutathione
(GSSG),
plasma
and
liver
homocysteine,
liver
S-adenosylhomocysteine
(SAH),
liver
methionine,
liver
S-adenosylmethionine
(SAM)
protein
concentrations
are
reported
in
nmol
per
mg
of
liver
sample.f.
The
level
of
hepatic
triglycerides
is
determined
by
using
L-Type
TG-M
Assay
Kit
according
to
the
manufacturer’s
instructions,
and
the
concentration
is
reported
in
mg
/
g
of
liver
sample.

X.
Western
BlotProteins
are
extracted
from
the
liver
and
analyzed
by
immunoblotting
using
horseradish
peroxidase-labeled
or
alkaline
phosphatase-labeled
secondary
antibodies.a.
Liver
tissues
are
collected
from
three
representative
mice
per
group
(based
on
liver
pathology
phenotypes).b.
Antibodies
against
actin,
heat
shock
protein
5
(HSPA5
formerly
GRP78),
DNA-damage
inducible
transcript
3
(DDIT3
formerly
CHOP),
betaine-homocysteine
methyltransferase
(BHMT),
and
sterol
regulatory
element
binding
transcription
factor
1
(SREBF1
formerly
NSREBP1)
are
used
as
primary,
while
IRDye680-
and
IRDye800-conjugated
antibodies
are
used
as
secondary.c.
Blots
are
scanned
using
the
Odyssey
system
and
intensity
of
the
bands
is
quantified
with
ImageJ.d.
The
intensity
of
protein
bands
on
the
blots
is
normalized
to
actin
and
to
corresponding
strain’s
high-fat
diet
(HFD)
samples.e.
Relative
protein
expression
of
BHMT,
DDIT3
(CHOP),
HSPA5
(GRP78),
and
SREBF1
(NSREBP1)
are
expressed
as
percent
of
control
HFD
mice.

XI.
RNA
isolation
and
gene
expression
analysis
a.
Total
RNA
is
extracted
from
liver
using
the
RNeasy
Mini
kit
according
to
manufacturer's
instructions.b.
RNA
concentrations
are
measured
with
NanoDrop
ND-1000
spectrophotometer.c.
RNA
quality
is
verified
using
the
Bio-Analyzer.d.
Total
RNA
(2
μg)
is
reverse
transcribed
using
random
primers
and
the
high
capacity
cDNA
archive
kit
according
manufacturer's
instructions.e.
The
following
primers
are
used
for
quantitative
real-time
Polymerase-Chain
Reaction
(qPCR)
according
to
Applied
Biosystems
protocol:

f.
Reactions
are
performed
in
a
96-well
assay
format
using
the
Roche
480
Thermocycler.g.
Each
plate,
containing
one
set
of
experimental
genes
and
a
housekeeping
gene
(Gusb),
are
plated
in
duplicate.
h.
The
cycle
threshold
(Ct)
for
each
sample
is
determined
from
the
linear
region
of
the
amplification
plot.i.
The
ΔCt
are
calculated
using
values
for
HFD_EtOH
mice
relative
to
strain-matched
control
HFD
means.j.
The
ΔΔCt
values
for
all
genes
relative
to
the
control
housekeeping
gene
Gusb
are
determined.k.
Relative
mRNA
abundance
is
expressed
as
percent
of
control
HFD
mice.